A double-stranded DNA-dependent protein serine/threonine kinase (DNA-PK) was purified from a nuclear extract of Raji Burkitt's lymphoma cells by a three-step column-chromatographic procedure. The main silver-stained band visualized after SDS/PAGE corresponded to an autophosphorylated polypeptide of about 350-kDa that represents the catalytic component. The existence of Ku DNA-binding protein as a regulatory component in the purified enzyme was revealed by Western blot/enzyme immunoassay and direct inhibition test with anti-Ku sera from the autoimmune patients. The DNA-PK catalyzed phosphorylation of synthetic peptides corresponding to Myc and RB proteins in a DNA-dependent manner, indicating that DNA-PK may recognize a second core-sequence motif Pro-Ser/Thr- in addition to the putative consensus sequences of -Ser/Thr-Gln. The level of enzyme activity was significantly higher in DMSO-induced G0/G1-arrested Raji cells as well as in the cells after release from DMSO than in the log-phase cells.